Abstract Cell-cell signaling pathways control the fate of cells during development and in adult stem cell contexts, and are frequently mis-regulated in disease states. Hedgehog, an ancient and highly conserved signaling pathway with many roles in animal development and homeostasis, controls the activity of Gli transcription factors, which then bind to sites within enhancers, or cis- regulatory elements, in the genome to influence the transcription of pathway target genes. Enhancers integrate spatial, temporal, physiological, and lineage information to control the pattern, timing, and levels of gene expression. Enhancer sequences account for a large proportion of disease-associated genomic variation in human populations. Investigating the mechanisms by which signal-regulated enhancers control gene expression helps us understand how organisms, tissues, and organs self-assemble during development, how they are maintained in adulthood, how they go awry in disease, and how new features are acquired and modified in the course of evolution. New tools for studying gene regulation at a detailed, mechanistic level have been developed over the past several years: these tools, combined with more established experimental approaches, provide opportunities to ask fundamental questions about how the regulatory genome functions and evolves. This highly collaborative project takes advantage of the remarkable conservation of the Hedgehog/Gli pathway with a multi-organismal approach, focusing on regulatory mechanisms that are common to fruit fly, chick, and mouse development, and benefiting from the experimental strengths of each of these model systems. The proposed project will combine fly and vertebrate model systems to accomplish three objectives: (1) Determine how Gli binding motifs in DNA, and common Gli motif variants, control the expression of Hedgehog target genes; (2) Investigate competition for occupancy of Gli binding motifs as a transcriptional regulatory mechanism; and (3) Decode the cis-regulatory logic of developmental Hedgehog/Gli target enhancers in flies and vertebrates.